PROJECT SUMMARY/ABSTRACT Leucine-Rich Repeat Kinase 2 (LRRK2) is a potential therapeutic target for Parkinson?s disease (PD) intervention. Mutations in LRRK2 are currently the most common genetic causes of PD, and several of these mutations directly or indirectly increase LRRK2 kinase activity. Furthermore, LRRK2 kinase activity has been implicated in its toxicity to neurons, and LRRK2 mutations may be pathogenic in other cell types such as microglia and those in peripheral tissues, causing symptoms associated with inflammation and gastrointestinal distress. The recent identification of endogenous LRRK2 kinase substrates as well as biochemical and genetic studies suggest that LRRK2 mutations cause dysfunction in a host of processes including autophagy, cytoskeletal dynamics, mitochondrial function, and synaptic transmission; however, the precise mechanisms by which LRRK2 mutants cause pathology are not understood. Elevation of oxidative stress is a major cellular mechanism of pathogenesis that has been associated with LRRK2, and recent evidence suggests that PD-associated mutations in LRRK2 increase levels of reactive oxygen species originating from damaged mitochondria. However, there are currently no tools to directly measure LRRK2 kinase activity in real-time in live cells in correlation with oxidative stress. Thus, in this project we will develop genetically-encoded fluorescent protein based sensors to overcome this technological barrier. Furthermore, these sensors will provide a key technology to directly analyze LRRK2 kinase activity in live cells, tissues, and animals, and they will be particularly well- suited for longitudinal experiments with respect to aging or drug treatments. The primary outcome of this project will be sensor technologies that are optimized for use by the research communities studying Parkinson?s disease, LRRK2, and oxidative stress.